Scrape cells off the plates and transfer to microcentrifuge tubes. Keep on ice.

Sonicate samples on ice three times for 5 seconds each.

Microcentrifuge for 10 minutes at 4°C, 14,000 x g, and transfer the supernatant to a new tube. If necessary, lysate can be stored at –80°C.

C. Immunoprecipitation

Take 200 μl cell lysate and add 10 µl of the immobilized antibody, incubate with rotation overnight at 4°C.

Wash pellet by using magnetic rack to pellet beads, then discard supernatant once completely clear and add 500 µl of 1X cell lysis buffer, vortex to resuspend and wash the beads, repeat 4 more times for a total of 5 washes.

Proceed to sample analysis by western blotting or kinase activity (section D).

For Analysis by Kinase Assay

Wash pellet by using magnetic rack to pellet beads, then discard supernatant once completely clear and add 500 Âµl of 1X kinase buffer, vortex to resuspend and wash the beads, repeat 1 more time for a total of 2 washes. Keep on ice during washes.

Source / Purification

Background

Epitope tags are useful for the labeling and detection of proteins using immunoblotting, immunoprecipitation, and immunostaining techniques. Because of their small size, they are unlikely to affect the tagged protein’s biochemical properties.